REVIEWpublished: 09 February 2018

doi: 10.3389/fnins.2018.00056

Frontiers in Neuroscience | www.frontiersin.org 1 February 2018 | Volume 12 | Article 56

Edited by:

Kim A. Staats,

University of Southern California,

United States

Reviewed by:

Gerardo Morfini,

University of Illinois at Chicago,

United States

Savina Apolloni,

Fondazione Santa Lucia (IRCCS), Italy

*Correspondence:

Rita Sattler

rita.sattler@dignityhealth.org

†Present Address:

Andrew T. Nelson,

Vickie and Jack Farber Institute for

Neuroscience, Department of

Neuroscience, Thomas Jefferson

University, Philadelphia, PA,

United States

‡These authors have contributed

equally to this work.

Specialty section:

This article was submitted to

Neurodegeneration,

a section of the journal

Frontiers in Neuroscience

Received: 13 October 2017

Accepted: 23 January 2018

Published: 09 February 2018

Citation:

Ghaffari LT, Starr A, Nelson AT and

Sattler R (2018) Representing Diversity

in the Dish: Using Patient-Derived in

Vitro Models to Recreate the

Heterogeneity of Neurological

Disease. Front. Neurosci. 12:56.

doi: 10.3389/fnins.2018.00056

Representing Diversity in the Dish:Using Patient-Derived in VitroModelsto Recreate the Heterogeneity ofNeurological Disease

Layla T. Ghaffari ‡, Alexander Starr ‡, Andrew T. Nelson † and Rita Sattler*

Department of Neurobiology, Barrow Neurological Institute, Dignity Health—St. Joseph’s Hospital and Medical Center,

Phoenix, AZ, United States

Neurological diseases, including dementias such as Alzheimer’s disease (AD) and

fronto-temporal dementia (FTD) and degenerative motor neuron diseases such as

amyotrophic lateral sclerosis (ALS), are responsible for an increasing fraction of worldwide

fatalities. Researching these heterogeneous diseases requires models that endogenously

express the full array of genetic and epigenetic factors which may influence disease

development in both familial and sporadic patients. Here, we discuss the two primary

methods of developing patient-derived neurons and glia to model neurodegenerative

disease: reprogramming somatic cells into induced pluripotent stem cells (iPSCs), which

are differentiated into neurons or glial cells, or directly converting (DC) somatic cells into

neurons (iNeurons) or glial cells. Distinct differentiation techniques for both models result

in a variety of neuronal and glial cell types, which have been successful in displaying

unique hallmarks of a variety of neurological diseases. Yield, length of differentiation, ease

of genetic manipulation, expression of cell-specific markers, and recapitulation of disease

pathogenesis are presented as determining factors in how these methods may be used

separately or together to ascertain mechanisms of disease and identify therapeutics for

distinct patient populations or for specific individuals in personalized medicine projects.

Keywords: IPSC, iNeuron, ALS, FTD, Alzheimer’s disease, Parkinson’s disease, Huntington’s disease

INTRODUCTION

Despite recent, rapid advancement in our understanding of biology and genetics, neurologicaldisease remains a persistent and fatal threat to human health. The World Health Organization(WHO) estimates that the fraction of worldwide deaths attributable to neurological conditionsincreased 89% from 2000 to 2015 (WHO, 2017). Alzheimer’s disease (AD) and other dementiasmake up the vast majority of these fatalities, but Parkinson’s disease (PD), epilepsy, Huntington’sdisease (HD), and motor neuron diseases such as amyotrophic lateral sclerosis (ALS) are eachresponsible for tens of thousands of deaths per year. The variety of symptoms and heterogeneity ofaffected individuals make diseases of the central nervous system (CNS) difficult to diagnose, study,and treat (Kim and Jeon, 2016; Van Cauwenberghe et al., 2016; Van Damme et al., 2017).

Ghaffari et al. Human in Vitro Models for Neurodegenerative Diseases

Staining of post-mortem tissue remains the only means ofofficially diagnosing several neurological diseases, and studyingthe mechanisms behind defining proteinopathies is difficultin this static state. The postmitotic nature of neurons anddifficulty of tissue access make biopsies of the CNS infeasible,and the blood-brain barrier isolates many biochemical signals ofdisease progression from peripheral biofluids. Thus, in order toproperly study pre-symptomatic disease or disease progressionmechanisms in patients, we must rely on human in vitromodels.

For genetically inherited disease, where a specific causalmutation has been identified, cellular models of disease canbe created by genetic manipulations (overexpression, siRNAknockout, CRISPR/Cas9 knock-in, or knock-out) of cells linesor primary rodent cell cultures. Alternatively, primary rodentcell cultures can be generated from existing animal modelsof disease (e.g., mutant SOD1 mouse model of ALS, R2/6mouse model of Huntington’s disease). These approaches docome with limitations. For example, overexpression models cansimultaneously mask subtle disease phenotypes while falselyexaggerating or creating entirely non-biological pathology.While these models may be used for early studies intodisease mechanisms, true biological representation, especiallyfor potential therapeutic development, requires endogenousexpression of known genetic mutations and their geneticinteractors, which emphasizes the need for patient-derivedmodels.

Patient-derived models become even more necessary forstudying sporadic disease, as there is no way of definitivelyrecapitulating the disease in a non-diseased organism.The genetic and epigenetic heterogeneity of sporadicpatients may make specific causative mutations statisticallyundetectable, or a confluence of small changes may be requiredto cause disease phenotypes. Such complexity would beimpossible to recapitulate through genetic manipulation ofa non-diseased cell, even if the full spectrum of contributoryexpression was identified. Since sporadic patients makeup the majority of patients in diseases such as ALS,Frontotemporal Dementia (FTD), and AD (Kim and Jeon,2016; Van Cauwenberghe et al., 2016; Van Damme et al.,2017), sporadic patient-derived cells offer the most widelyapplicable tool for studies of disease mechanisms and therapeuticscreens.

Two primary methods for generating patient-derived cellshave emerged. Somatic cells can be reprogrammed into inducedpluripotent stem cells (iPSCs) and then differentiated into thedesired cell type or directly converted (DC) into the desired celltype (Figure 1). Each method has been expanded over the lastdecade for differentiation into a wide variety of CNS cells usedto study and treat neurological disease. Here, we review howthese models have developed over the last 10 years, the methodsused to differentiate them, the specific cell types those methodsproduce, and how disease has been studied in these models. Wealso highlight how human culture models are providing us withnew mechanistic insights that were previously unattainable withpostmortem patient tissue analysis or animal models of disease,and discuss the current limitations of in vitro modeling of CNSdisease.

IPSC OVERVIEW

Early experiments in using stem cells to model neurons werelargely developmental and relied upon the differentiation ofembryonic stem cells (ESCs) (Thomson et al., 1998). Becausethese cells were not patient-derived, they were either culturedfrom mutant mouse embryos (Di Giorgio et al., 2007) orwere healthy ESCs transfected to overexpress known mutants(Karumbayaram et al., 2009). These models encounter the samesetbacks as all over-expressions, and thus offer little improvementover simpler models. Recently, preimplantation genetic diagnosis(PGD) has offered an opportunity for ESCs to be generated fromembryos known to carry disease causing mutations, namely thehexanucleotide expansion of C9orf72 (C9) (Cohen-Hadad et al.,2016). Methylation analysis of motor neurons differentiated fromthese ESCs compared to adult C9 patient-derived iPSCs revealedhypermethylation of the iPSCs, which would suggest that ESCsprovide a better representation of patient epigenetics. However,ethical concerns, the limited availability of endogenouslymutatedESCs, and their inability to be used tomodel sporadic disease callsfor the ability to generate human-derived in vitro models fromadult patients. Experiments which showed that ESC fusion couldconfer pluripotency to fibroblasts suggested that certain cellularproducts might induce pluripotency even in an adult somatic cell(Cowan et al., 2005).

Since the initial, groundbreaking discovery of fourtranscription factors (Oct3/4, Sox2, c-Myc, and Klf4) capable ofconverting adult mouse (Takahashi and Yamanaka, 2006) andhuman (Yu et al., 2007) fibroblasts into pluripotent stem cells, anumber of new methods for reprogramming somatic cells havebeen developed, including the use of various viral and chemicaltransfection methods to express optimized pluripotency factors.iPSCs can be generated from a wide variety of somatic cells,including skin, peripheral blood mononuclear cells, and hair,which enables non- or minimally-invasive collection of patientsamples to generate models of their disease (Egusa et al., 2010).

IPSC DIFFERENTIATION METHODS

Prior to the choice of which specific neural cell to generate,the basic method of differentiation must be chosen. Manyprotocols rely on the addition of small molecules and growthfactors to first induce embryoid bodies followed by neuralrosettes (Elkabetz et al., 2008), or procede directly from iPSCsto neuroprogenitor cells (NPCs) via dual-SMAD signalinginhibition (Chambers et al., 2009). NPCs can be maintained andfurther differentiated and matured using growth factors. NPCscan be infinitely expanded, frozen down, and thawed to provide amid-differentiation starting point, which reduces derivation timeby∼30% (Brafman, 2015).

Some differentiation protocols combine small molecules andgrowth factors with transfection of lineage-specific transcriptionfactors, in a manner similar to direct conversion. For example,Wen et al. used transcription factors established by Son et al.for direct conversion to generate iPSC induced motor neuronsfor the study of repeat associated non-ATG (RAN) translateddipeptide repeat proteins (DPRs) in C9 ALS (Son et al., 2011;

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FIGURE 1 | Illustration of the generation and application of patient-derived models. Patient cells are used to generate iPSC or DC-based in vitro models of familial or

sporadic disease. These models are studied to elucidate disease-contributing mechanisms and screen preclinical therapeutics, which can be translated into

treatments for donor patient populations.

Wen et al., 2014). Other groups have reported that transcriptionfactors may be used to more quickly generate very pure culturesof dopaminergic and GABAergic neurons (Theka et al., 2013;Yang et al., 2017). Neural progenitor cells (NPCs) have beenderived using only small molecules (Reinhardt et al., 2013), and Liet al. discovered that microRNA 199a could induce angiogenesisin iPSCs, which may indicate that small-molecule only andmicroRNA based differentiation protocols, which have been usedmore widely in direct conversion, could be developed for iPSCs(Li Z. et al., 2015).

Another differentiation alternative attempts to recreate thephysics of the biological environment by differentiating iPSCsin structured organoids. Unique basal hydrogels, custom culturechambers, and physical agitation can be used to encourageiPSCs to differentiate into a specific three-dimensional structure(Lindborg et al., 2016). This may lead to greater representationof brain-layer formation, which may improve cell maturity,stimulate expression of disease phenotypes, and provide a moreaccurate model for drug screens (Raja et al., 2016). In addition,organoids can provide a method of modeling diseases which are

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difficult to recreate in animal models, such as microencephaly(Lancaster et al., 2013).

IPSC DIFFERENTIATED CELL TYPES

Human iPSCs have been successfully differentiated into anumber of disease-relevant cell types, as verified by morphology,gene expression profiles, and cell-specific protein expression.Many differentiation protocols begin with the development of(NPCs). These have been used primarily for the development oftherapeutic neuron replacement (Nicaise et al., 2017). Similarly,oligodendrocyte precursors have been used to drive implantedcells to an oligodendrocyte fate for remyelination therapies(Douvaras et al., 2014; Kawabata et al., 2016). Differentiationbeyond precursors yields neurons and glia with varyingefficiencies and purity. Variations in small molecule and growthfactor selection, maturation duration, and culture conditionscontinue to be optimized.

iPSC Brain-Native Neuronal SubtypeDifferentiationiPSCs have been differentiated into a number of brain-nativeneuronal subtypes. Cortical glutamatergic neurons can begenerated by initializing neuralization using retinoids and SMADinhibition. This generates cerebral cortex stem cells, whichmature over 60 days into neurons representing the corticallayers (Shi et al., 2012). Viral expression of LMX1A in NPCcultured on PA6 stromal cell yields dopaminergic (DA) neurons(Sanchez-Danes et al., 2012). The combined effect of virallyexpressed ASCL1, NURR1, and LMX1A can generate iPSC DAneurons without the need for PA6 co-culture (Theka et al., 2013).Inhibitory GABA-ergic neurons can also be derived via viralinduction of ASCL and DLX2 (Yang et al., 2017). These are buta few examples of the wide variety of neuronal subtypes createdfrom iPSCs from an even wider array of protocols.

iPSC Spinal Motor Neuron DifferentiationNeurons native to the spinal cord and periphery, includingmotorand sensory neurons, can be generated readily from iPSCs. iPSCmotor neurons (iPSCMNs) are generated by treatment withretinoic acid and sonic hedgehog (SHH), which is required forfloor-plate differentiation (Ericson et al., 1996). iPSCMNs expressHB9, Islet-1, and ChAT, which indicate a mature cholinergicmaturation (Dimos et al., 2008). Peripheral nerves have alsobeen successfully modeled using iPSCs. Nociceptor precursorcells can be generated from stem cells in 24 days, transfectedto overexpress Neurog1, and matured into capsaicin-sensitivenociceptors to study nerve pain disorders (Boisvert et al., 2015).

iPSC Glial DifferentiationGlia can also be recreated from iPSCs, including astrocytes,oligodendrocytes, and microglia. These can be studied asmono-cultures or co-cultured with neurons to study non-cellautonomous effects on disease, be they contributory orcompensatory.

Astrocytes are necessary to provide neurotrophic supportand maintain healthy synaptic conditions. Krencik et al first

reported the differentiation of iPSCs into astrocytes (Krenciket al., 2011). The resulting cells were GFAP positive, S100Bpositive, and took 180 days to differentiate, because astrocytedifferentiation occurs after neuronal differentiation (Emdadet al., 2012). Differentiating iPSCs into neural stem cells beforeastrocyte differentiation reduced the generation time to 6weeks (Shaltouki et al., 2013). Fluorescence-activated cell sorting(FACS) of differentiated iPSCs virally transfected to express GFPunder a GFAP promoter allows for the selection of a purelyastrocytic culture and reduces “noise” from neurons and otherglia remaining from early periods of differentiation (Zhang et al.,2016).

In addition to the use of oligodendrocyte precursors(OPCs) in therapeutic transplantation, mature iPSC-derivedoligodendrocytes have been generated. Early reports suggestedthat iPSCs might contain some inherent factor which preventedoligodendrocyte maturation (Tokumoto et al., 2010), however, alow yield, high purity, three-month protocol was soon developed(Hu et al., 2009). These mature oligodendrocytes were capableof generating myelin sheaths. Oligodendrocyte precursors can bematured by co-culture with dorsal root ganglion (DRG) neuronsand will myelinate them in vitro (Czepiel et al., 2011). Douvaraset al. used a protocol similar to iPSMN differentiation, includingadherent dual SMAD inhibition and early caudalization andventralization, to increase OPC yield and shorten differentiationtime to 75 days (Douvaras et al., 2014).

Microglia derive from macrophages but do not gain certaindistinct features until they develop in concert with the nervoussystem. Early attempts to generate microglia from iPSCs resultedin cells called microglia-like macrophages, and while they didnot express typical microglial morphology, they were positive forIBA1, CD45 and CD11b (Muffat et al., 2016). When microglia-like macrophages were co-cultured with iPSCNs, they exhibiteda more mature microglial morphology and phagocytic behavior(Haenseler et al., 2017a). Recently, Douvaras et al. reported amethod to develop mature, ramifying microglia without co-culturing (Douvaras et al., 2017).

iPSC Differentiation into Neural-InterfacingCell TypesNon-neuronal cells relevant to neurological disease have alsobeen derived. iPSCs can be differentiated into myoblasts,which form contracting, electrically and chemically responsivemyotubes that are capable of forming neuromuscular junctions(NMJs) (Demestre et al., 2015). Additionally, endothelial cellscan be generated to model the blood-brain-barrier, which issuggested to be compromised in neurodegeneration (Lippmannet al., 2012).

With the ability to differentiate into the full complementof neurons, glia, and neural-interfacing cells, iPSCs provide apowerful model for the study of disease in individual cells, inco-culture, and in three-dimensional reconstructions of complextissues. Many examples of subtype-specific differentiationmethods focus on the expression of specific marker proteins.While this is an important indicator of an individual cell type,more parameters can and should be considered when evaluating

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differentiation protocols. For example, positive expression ofmarker proteins should be correlated with the lack of expressionof specific marker proteins of similar, yet distinct, cell types, e.g.,cortical forebrain neurons should lackmarkers of motor neurons.In addition, gene expression profiles should be compared todata from either primary murine cultures of the same cell typeor isolated cell types from postmortem brain tissues. Similarly,morphology and electrophysiological activity should be includedin the cell characterization. This will increase confidence inclaims of cell-type specificity and also provide more experimentalreadouts to use as standards for maintaining consistent modelsacross multiple rounds of differentiation.

Additional steps may be employed to reduce differentiationvariability and to ensure disease relevance of observed deficits.While the use of multiple disease and control cell lines controlsfor the variability between individual human subjects, multipledifferentiations of the same cell line accounts for variabilitybetween differentiations. In addition, CRISPR-Cas9 mediatedgene editing allows for the correction of disease-causingmutations (Cong et al., 2013). This provides the possibilityof creating isogenic control iPSC lines, which geneticallyand epigenetically match patient derived lines, except for thecorrected mutation. If this correction alleviates deficits, itsupports the association between the gene mutation-dependentphenotypes and actual disease pathology. This can bestrengthened further by using the same technique to introduceknown mutations in healthy control iPSCs, creating a positivecontrol. Unfortunately, isogenic controls cannot be created tostudy sporadic disease which, as mentioned above, represents themajority population of most neurodegenerative diseases.

IPSC MODELS OF DISEASE

Amyotrophic LateralSclerosis/Frontotemporal DementiaiPSCs have been used extensively to model neurological disease(Figure 2). ALS, a fatal motor neuron disease, and FTD,a neurodegenerative dementia, appear to exist on a geneticspectrum of disease, with several shared genetic causes andidentifying pathologies. It is in this spectrum that the widestvariety of iPSC modeling has been employed. iPSCMNs arethe most widely used, and have been shown to demonstratethe specific pathology and physiology of ALS subtypes. Dimoset al. were the first to report the generation of iPSCMNsfrom an ALS patient that expressed the motor neuron-specifictranscription factors HB9 and Islet 1 (Dimos et al., 2008). Itwas not until four years later that actual disease phenotypeswere first shown in TDP-43 ALS patient-derivedmotor neurons.Egawa and colleagues differentiated iPSCMNs from threeALS patients carrying three distinct TDP-43 mutations andshowed that, among other deficits, the diseased motor neuronsexhibited hallmark TDP-43 aggregates similar to those foundin postmortem ALS brain tissues (Egawa et al., 2012). Theauthors were able to rescue these phenotypes with a histoneacetyltransferase inhibitor, providing first evidence that patient-derived iPSCMN culturemodels could be used for drug screening

purposes. iPSC modeling of FTD soon followed when Almeidaand colleagues generated iPSC neurons and microglial cellsfrom FTD patients carrying a mutation in progranulin (PGRN)(Almeida et al., 2012). The differentiated human cells confirmedthe presence of PGRN haploinsufficiency, in addition to aidingin the new discovery of mutant PGRN-specific deficits of certainprotein kinase pathways. When the C9orf72 hexanucleotideexpansion was discovered as the most common shared mutationin ALS/FTD disease spectrum in 2011(DeJesus-Hernandez et al.,2011; Renton et al., 2011), the fastest way to study diseasemechanisms was via iPSCs, as the generation of the repeatexpansion in mouse models proved to be difficult. Severallaboratories shortly thereafter provided the first evidence ofthe three major disease mechanisms of mutant C9orf72 usingiPSC neurons: haploinsuffiency, RNA foci formation, and repeat-associated non-ATG (RAN) translation (Almeida et al., 2013;Donnelly et al., 2013; Lagier-Tourenne et al., 2013; Sareenet al., 2013). These studies also revealed disease-mediatedaberrant gene expression and neuronal function in patientiPSCs. Numerous follow up studies examining both neuronaland glial iPSC culture models have since been used to furtherinvestigate how mutant C9orf72 leads to neuronal degeneration,ranging from studies on nucleocytoplasmic trafficking deficits(Freibaum et al., 2015; Zhang et al., 2015; Lopez-Gonzalezet al., 2016; Westergard et al., 2016) to the toxic potentialof RAN dipeptides, as recently reviewed (Selvaraj et al.,2017).

Other familial ALS mutation phenotypes, such as valosin-containing protein (VCP) (Hall et al., 2017), TAR DNA-bindingprotein 43 (TDP-43) (Serio et al., 2013; Barmada et al., 2014), andsuperoxide dismutase (SOD1) (Bhinge et al., 2017), have beenrecapitulated in iPSCs showing characteristic proteinopathies,and endoplasmic reticulum (ER) and oxidative stress. Todetermine which disease pathologies in the model are causedprimarily by the mutation, isogenic corrections of known geneticcauses, such as SOD1, have been generated, and were shown tosuccessfully rescue the mutant phenotypes, such as previouslydescribed hyperexcitability in SOD1 mutant iPSCMNs (Waingeret al., 2014; Bhinge et al., 2017).

Importantly, considering sporadic disease represents 90%of ALS patients, sporadic iPSCs have also been differentiatedinto neurons and were shown to exhibit TDP-43 pathology(Burkhardt et al., 2013; Qian et al., 2017). Furthermore, geneexpression profiling of sporadic ALS iPSCMNs suggested deficitsin mitochondrial function (Alves et al., 2015). More studiesare required to better understand the pathogenesis of sporadicdisease, and human cell culture models are currently the only wayto gain insight into what triggers and propagates sporadic ALSand FTD.

Limited studies have addressed the role of glial cells inALS/FTD, although both ALS and FTD patient-derived iPSCastrocytes have been employed to study the non-cell autonomousmechanisms of neurodegeneration. Interestingly, while studiesusing SOD1 mutant mouse models and primary culturesgenerated from these mice unequivocally demonstrated thatmutant astrocytes trigger motor neuron degeneration, iPSCastrocytes differentiated from ALS patients carrying a TDP-43

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FIGURE 2 | Examples of patient-derived in vitro models of neurological disease. Select examples of how patient-derived iPSC and DC neurons, astrocytes, and

microglia have been used to model ALS/FTD, AD, HD, and PD. Numbers correspond to the following references: 1. Donnelly et al., 2013; 2. Wang et al., 2014; 3.

Burkhardt et al., 2013; 4. Wen et al., 2014; 5. Hall et al., 2017; 6. Serio et al., 2013; 7. Barmada et al., 2014; 8. Qian et al., 2017; 9. Almeida et al., 2012; 10. Liu et al.,

2016; 11. Lim et al., 2016; 12. Son et al., 2011; 13. Su et al., 2014; 14. Meyer et al., 2014; 15. Yagi et al., 2011; 16. Kondo et al., 2013; 17. Muratore et al., 2014; 18.

Balez et al., 2016; 19. Sproul et al., 2014; 20. Jones et al., 2017; 21. Zhao et al., 2017; 22. Abud et al., 2017; 23. Hu et al., 2015; 24. Hou et al., 2017; 25. Nekrasov

et al., 2016; 26. Grima et al., 2017; 27. Mattis et al., 2015; 28. Juopperi et al., 2012; 29. Hsiao et al., 2014; 30. Hsiao et al., 2015; 31. Liu et al., 2013; 32. Devine

et al., 2011; 33. Imaizumi et al., 2012; 34. Seibler et al., 2011; 35. Haenseler et al., 2017b; 36. Puschmann et al., 2017.

mutation, while exhibiting TDP-43 mislocalization themselves,did not affect motor neuron survival in a co-culture model (Serioet al., 2013). On the other hand, iPSC astrocyte-conditionedmedia derived from C9orf72 patients leads to impairments ofiPSC motor neuron autophagy (Madill et al., 2017). Similarly,iPSC astrocytes from mutant VCP patients affect motor neuronsurvival in vitro (Hall et al., 2017). In addition, transplantingsporadic ALS iPSCs into the spinal cord of WT mice resultedin differentiation into astrocytes and triggered a loss of neuronsin the spinal cord, with accompanying movement deficits (Qianet al., 2017).

Even fewer studies to date have generated and characterizediPSC-derived oligodendrocytes. Ferraiuolo and colleagues usedboth sporadic and fALS patient-derived oligodendrocytes toshow that, similar to astrocytes, diseased oligodendrocytesaffect motor neuron survival (Ferraiuolo et al., 2016). At thesame time, Livesey and colleagues studied the maturation andelectrophysiological properties of C9orf72 patient-derived iPSColigodendrocytes and did not observe any obvious impairmentsor alterations of these cells when grown in mono-cultures(Livesey et al., 2016).

The only study describing iPSC microglial cells fromALS or FTD patients was reported by Almeida et al. who

showed that PGRN FTD patient microglial cells exhibitprogranulin haploinsufficiency, but do not show the deficits inserine/threonine kinase S6K2 observed in iPSC neurons from thesame patients (Almeida et al., 2012).

Alzheimer’s DiseaseThe most common dementia, Alzheimer’s disease (AD), has beenexamined extensively in patient-derived iPSCs, and these studiesbeen thoroughly reviewed (Devineni et al., 2016; Arber et al.,2017; Robbins and Price, 2017; Tong et al., 2017). One of the firstreports using patient-derived iPSC neurons to study mechanismsof AD examined the pathogenesis of familial Presenilin 1 (PS1)and Presenilin 2 (PS2) (Yagi et al., 2011). The mutant iPSCneurons exhibited increased amyloid β 42 secretion, which wasinhibited by a γ-secretase inhibitor. This study was followedby a report from Israel and colleagues who found an increasein Aβ and phosphorylated tau (p-tau) in familial amyloid-βprecursor protein (APP) duplication (APP(Dp)) and sporadiciPSC cortical neurons (Israel et al., 2012). Treating patientneurons with β-secretase inhibitor, but not γ-secretase inhibitors,significantly reduced the documented disease phenotypes in thecells, providing new knowledge on the mechanisms of APPpathogenesis.

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These early studies were followed by reports using bothfamilial and sporadic AD patient iPSCs differentiated intocortical neurons or, to a limited degree, glial cells to betterunderstand mechanisms of neurodegeneration in AD and fornovel therapeutic target investigation. For example (Duan et al.,2014), used sporadic AD patient iPSCs with ApoE3/E4 genotypesand reported not only an increased ratio of Aβ42/40 andresponsiveness to γ-secretase inhibitors, but also increasedsusceptibility to glutamate toxicity with a concomitant increasein free intracellular calcium levels, suggesting that actualneuroprotection could be examined using these patient-derivedneurons. Similarly, iPSCs from AD patients carrying an APPmutation (V717I) showed increased expression levels of APPand Aβ, in addition to aberrant β and γ secretase cleavageof APP and increased levels of total and phosphorylated tau(Muratore et al., 2014). Interestingly, the authors were able toshow that treatment with specific Aβ antibodies early duringthe differentiation process rescued the mutant APP phenotypes.This new paradigm of testing drugs in patient-derived cells wasstrengthened by a study in which apigenin has been tested as apotential anti-inflammatory therapeutic in AD patient-derivedneurons co-cultured with activated murine microglial cells. Thetreatment was successful in reversing morphological deficiencies,reducing hyper-excitability, and protecting against apoptosis(Balez et al., 2016).

Sproul and colleagues studied iPSC derived neural precursorcells (NPC) to understand whether, even at a pre-neuronalstage of development, human cells show AD phenotypes (Sproulet al., 2014). Differentiating iPSCs from mutant PS1 patients intoNPCs, the authors observed an increased Aβ42/40 ratio and anaberrant gene expression profile after transcriptome analysis. Aselect number of gene aberrations were confirmed to be similarlyaltered in postmortem AD brain tissues. Similar findings wereconfirmed in PS1 patient iPSC neurons (Mahairaki et al., 2014).Interestingly, generating isogenic iPSC lines using TALENs (TALEffector Nucleases) technology to insert PS11 E9 mutations inotherwise healthy control patient lines lead to the discovery thatthis mutation represents a toxic gain of function mutation thatimpairs PS1-dependent γ secretase activity, but not unrelated γ

secretase functions (Woodruff et al., 2013).Few studies thus far have used patient-derived iPSC astrocytes

to model Alzheimer’s disease. Kondo et al showed that iPSCastrocytes from sporadic AD patients and patients with an(APP)-E693delta mutation displayed ER and oxidative stress,which was alleviated with treatment of docosahexaenoic acid(DHA) (Kondo et al., 2013). A more recent study showedthat iPSC astrocytes from familial and sporadic AD patientsdisplay significant morphological phenotypes with an overallatrophic profile andmislocalization of astroglial marker proteins,including S100B (Jones et al., 2017). IPSCs astrocytes fromnormal individuals with APOE ε4/ε4 genotypes were lesseffective in promoting neuronal survival and synaptogenesiswhen compared to iPSC astrocytes from normal individualswith APOE ε3/ε3 genotypes (Zhao et al., 2017), illustrating thatpatient-derived culture models could aid in exploring the roleof individual apoE isoforms in AD disease pathogenesis. Finally,iPSC astrocytes differentiated from patients carrying the PSEN1

delta E9 mutation exhibited typical AD pathology—increased β

amyloid, altered cytokine release, defective calcium homeostasis,increased oxidative stress and reduced lactate secretion (Oksanenet al., 2017). None of these phenotypes were present in healthycontrol or gene-corrected isogenic iPSC astrocytes.

Similar to ALS above, only one study to date has usedpatient-derived microglial cell to study AD disease mechanisms.Abud and colleagues generated iPSC microglial-like cells andshowed altered gene expression in response to Aβ fibrils andaberrant phagocytic activity when exposed to brain-derivedtau oligomers (Abud et al., 2017). Further work is requiredto determine whether the observed AD iPSC-derived glialphenotypes contribute to the neuronal disease pathogenesis in anon-cell autonomous manner.

To overcome the observed inability of 2 dimensional iPSCneuron cell culture models to generate extracellular proteinaggregations, the AD field has quickly tried to model AD in 3dimensional (3D) human cell culture systems (reviewed by andChoi et al., 2016; Lee et al., 2017). One of the first publishedstudies using 3D iPSC differentiated neuron cultures showed thatonly in the 3D environment did the AD neurons display p21-activated kinase-mediated sensing of Aβ oligomers, in additionto the presence of F-actin associated protein phenotypes (Zhanget al., 2014). Choi and colleagues developed a 3D iPSC neuronculture model from familial AD patients (mutant APP andmutant presenilin), which, for the first time, represented a diseasemodel that displayed hallmarks of AD pathology: extracellulardeposition of amyloid-β, including amyloid-β plaques, andaggregates of phosphorylated tau, as well as filamentous tau (Choiet al., 2014; Kim et al., 2015). More recent studies have appliedthe 3D culture model to generate high throughput models fordrug screening against tau aggregation (Medda et al., 2016),or to compare efficacy of drug candidates (β or γ secretaseinhibitors) in 2D vs. 3D culture systems (Lee et al., 2016).Finally, the generation of brain organoids, initially developedto model human brain development (Lancaster et al., 2013),has been adopted to study mechanisms of AD as well. Rajaand colleagues reported that brain organoids from familial ADpatients recapitulate AD disease phenotypes and pathologiesincluding amyloid aggregation, hyperphosphorylated tau, andendosome abnormalities, all of which were reduced by treatmentwith secretase inhibitors (Raja et al., 2016).

Huntington’s DiseaseZhang and colleagues were the first group to characterize HDpatient-derived iPSC neuronal cells (Zhang et al., 2010). Theauthors differentiated iPSCs into striatal neurons expressing cell-specific markers such as DARPP-32. The neurons maintainedthe CAG repeat expansion and showed increased susceptibilityto growth factor removal as shown by enhanced caspaseactivity. The same research team also generated the first gene-corrected isogenic cell lines, which resulted in the rescue ofdisease phenotypes, including the susceptibility to cell death andmitochondrial abnormalities (An et al., 2012). A large consortiumof Huntington’s disease researchers reported increased cell death,sensitivity to stressors, increased glutamate toxicity, and reduced

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sporadic electrical firing in HD patient-derived iPSC striatal-like neurons as characterized by the expression of Map2a/band Bcl11B (iPSC Consortium, 2012). Interestingly, the severityof several of these phenotypes increased in proportion tothe patient’s CAG-expansion length in the HTT gene. Similarfindings were reported by Mattis and colleagues, who reportedincreased cell death following BDNFwithdrawal, but also showedthat increased susceptibility to glutamate toxicity could beblocked by NMDA and AMPA receptor inhibitors (Mattis et al.,2015), suggesting novel therapeutic approaches for HD.

Several other potential Huntington’s disease pathways havebeen identified using patient derived iPSC neurons, such asmiR196a dysregulation (Cheng et al., 2013), MAPK and Wntsignaling (Szlachcic et al., 2015), the protective potential ofA2A adenosine receptor activation (Chiu et al., 2015), andastrocyte-mediated cytokine-induced neuronal cell death (Hsiaoet al., 2014). CRISPR-Cas9 gene-corrected isogenic HD patientlines showed rescue of HD phenotypes, such as deficits inmitochondrial respiration (Xu et al., 2017). Most interestingly,though, these isogenic lines showed that gene expressiondifferences between HD and healthy control iPSCs were notpresent when HD lines were compared to their isogenic control,suggesting that general differences in genetic backgroundcould be falsely identified as transcriptomic abnormalitiestriggered by the disease-causing mutation. Neskarov et al.reported that HD iPSC neurons show increased numbers oflysosomes/autophagosomes and exhibit increased cell deathalongside nuclear indentation (Nekrasov et al., 2016). Furthernuclear deficits, including nucleoporin aggregation and impairednucleo-cytoplasmic transport, have recently been demonstratedin HD patient-derived neurons (Grima et al., 2017); similarto what has been described in ALS (Kim and Taylor, 2017).One of the most recent studies showed that previously reportedrepression of peroxisome proliferator-activated receptor delta(PPAR-δ)—a ligand-gated transcription factor that promotesmitochondrial biogenesis and oxidative metabolism—in an HDmouse model is also found in HD patient-derived iPSC neurons(Dickey et al., 2016, 2017). Indirect activation of PPARγ via thesmall molecule compounds bexarotene or KD3010 significantlyrescued impaired oxidative metabolism in HD neurons.

There have been few reports of iPSC differentiations intonon-neuronal cells for HD. Huntington’s iPSC astrocytes wereshown to express higher levels of vascular endothelial growthfactor-A (VEGF-A) (Hsiao et al., 2015) and formed electron-clear vacuoles, which increased during the differentiation periodbut appeared independently of cellular stressors (Juopperi et al.,2012). To date, patient derived microglia and oligodendrocyteshave not been studied in Huntington’s disease, but differentiationof HD iPSCs into brain microvascular endothelial cells revealedabnormalities in angiogenesis and blood brain barrier properties(Lim et al., 2017).

In summary, the use of patient-derived iPSC cultures inHD has provided new insight regarding disease pathogenesisand also provided a novel model to perform pre-clinicaldrug discovery (Tousley and Kegel-Gleason, 2016). A potentialadditional application of these patient-derived cells lies inthe transplantation of stem cells or their derivatives to

replace diseased or lost neurons. While not discussed in thisreview, numerous efforts have been made toward successfultransplantation of cells at the iPSC stage or the neural precursorstage in rodents with the hope to create a therapeutic path towardHD patient transplantations (Golas and Sander, 2016; Connor,2017; Choi et al., 2018).

Parkinson’s DiseaseParkinson’s disease (PD) has been extensively modeled usingpatient-derived iPSCs (Cobb et al., 2017). PD is modeled chieflyby the differentiation of iPSCs into dopaminergic (DA) neurons.Park et al. created the first Parkinson’s disease iPSC line, togetherwith other neurodegenerative disease lines (Park et al., 2008).Patient-derived DA neurons exhibit the typical pathology of PD:the presence of Lewy bodies and their main component, α-synuclein. This pathology is present in iPSC DA neurons frompatients with mutations in the SNCA gene and in patients withmutations in leucine-rich repeat kinase 2 (LRRK2) (Devine et al.,2011; Nguyen et al., 2011) and, rarely, in patients with mutationsin parkin (Imaizumi et al., 2012), but not in patients with amutation in PTEN-induced putative kinase 1 (PINK1) (Jianget al., 2012). This reflects the pathology as it has been observedin postmortem patient brain tissue. Other disease phenotypespreviously observed in either mouse models or autopsy tissuewere confirmed in patient-derived familial and sporadic iPSCDAneurons, including mitochondrial deficits and oxidative stress(Byers et al., 2011; Cooper et al., 2012; Hsieh et al., 2016) andlysosomal and autophagy deficits (Mazzulli et al., 2011, 2016;Sanchez-Danes et al., 2012; Schondorf et al., 2014; Fernandeset al., 2016).

Similar to the aforementioned neurodegenerative diseases,researchers are now using genetically corrected patient-derivediPSCs to study PD pathogenesis in order to reduce the potentialfor general patient-to-patient genetic variability to obscuredisease-specific cellular and genetic alterations, and to pinpointspecific disease mechanisms linked to the genetic mutationexamined, and not to potential cellular deficits arising from theactual cell differentiation process (Soldner et al., 2011; Arias-Fuenzalida et al., 2017; Qing et al., 2017).

Parkinson’s disease has not been studied extensively in iPSC-derived glia; however, Haenseler et al. have recently shownthat phagocytosis is impaired in SNCA triplication microglia(Haenseler et al., 2017b).

Neuroinflammation and NeuropsychiatricDiseasesModeling disease with iPSCs extends beyond neurodegeneration.Although much research in de-myelinating disease, such asmultiple sclerosis (MS), focuses on differentiating healthyoligodendrocyte precursors for transplantation replacementtherapy (Kawabata et al., 2016; Sato et al., 2017), MS patient-derived iPSCs have been differentiated into neurons (Song et al.,2012), astrocytes (Song et al., 2012), oligodendrocyte precursors(Nicaise et al., 2017), and oligodendrocytes (Song et al., 2012;Douvaras et al., 2014). The only differences so far identified arean inability of MS iPSCNs to fire spontaneous action potentials(Song et al., 2012), and reduced neuroprotection by MS iPSC

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oligodendrocyte precursors (Nicaise et al., 2017). Microglia,which have been sparingly used to model neurological disease,have also been used to model Rhett syndrome (Muffat et al.,2016). The use of iPSCs to model neuropsychiatric disorders isexpanding rapidly, as reviewed by Ho et al. (2015).

Reprogrammed patient-derived cells have been used to modelsporadic and familial neurodegenerative, neurodevelopmental,and neuropsychiatric disease. Much early work using iPSCsto study neurological disease focused on validating previously-observed phenotypes from animal models and postmortemautopsy tissues. Now that the ability of iPSC-derived CNSmodels to recreate disease pathology has been established, morerecent studies are beginning to use iPSCs to tease out diseasemechanisms and screen for pre-clinical therapeutics. Theseefforts are supported by continuing improvements in cell type-specific differentiation methods, the expansion of co-culture and3D culture models, and the generation of genetically correctedisogenic patient lines to account for the diversity of patients’intrinsic genetic backgrounds.

Despite the advantages and advancements of patient-derivediPSCs, concerns about differentiation time, culture specificityand purity, teratoma formation in transplantation, and, mostimportantly, the difficulty for rejuvenated cells to model agingdiseases has led to the development of a complimentarymethodology: lineage-specific direct conversion of patient-derived somatic cells.

DIRECT CONVERSION OVERVIEW

One distinct advantage of direct lineage conversion, also knownas transdifferentiation, is that there is no reprogrammingstep. The epigenetic identity of the starting cells remainsafter conversion, as opposed to the hypermethylation observedwhen somatic cells are rejuvenated into iPSCs (Cohen-Hadadet al., 2016). This retains “cellular memories.” Most conversionprotocols feature mesoderm to ectoderm conversions—meaningfibroblasts to neurons. Due to the infancy of this field, thedefinition of an induced neuron (iN) has many meanings (Yanget al., 2011). Direct conversion can occur from fibroblasts(Vierbuchen et al., 2010), blood (Lee et al., 2015), urine cells(Zhang S. Z. et al., 2016), hepatocytes (Marro et al., 2011), andadipocyte progenitors (Yang Y. et al., 2013), which providesa wide variety of non-invasive starting materials for modelingpatient disease.

The first report of direct lineage reprogramming byVierbuchen et al. (2010) used combinatorial expression ofthree neural lineage-specific transcription factors—Brn2, Ascl1,and Myt1L, known as the BAM factors—to induce neurons frommouse fibroblasts (Vierbuchen et al., 2010). The induced neurons(hereafter called iNeurons or iNs) that result from this methodexpress pan-neuronal markers including Tuj1, NeuN, MAP2,and synapsin. These iNs also fire induced and spontaneousaction potentials and form functional synapses. However, theBAM factors alone cannot convert human fetal or postnatalfibroblasts into functional neurons. The addition of the basichelix-loop-helix transcription factor NeuroD1 to the BAM factor

conversion protocol was found to be necessary for iNs to beproduced from human somatic cells (Pang et al., 2011).

Here, we will first describe the general methods used todirectly convert somatic cells. We then specify conversionmethods applied to the generation of neurons and glial cells,followed by a summary on how these directly converted cells havebeen studied in varying neurodegenerative diseases thus far.

DIRECT CONVERSION METHODS

There are three primary methods of directly converting humansomatic cells into CNS cells. Transcription factor overexpression,microRNA overexpression, and small molecule treatments can,individually or in concert, generate a variety of cell types fromthe aforementioned starting materials.

Transcription Factor-Mediated DirectConversionSince Pang et al. first successfully created human iNs, a numberof other groups have obtained similar results using some or all ofthe BAM transcription factors (Ambasudhan et al., 2011; Caiazzoet al., 2011; Pfisterer et al., 2011; Son et al., 2011). Furthermore,Marro et al. used the BAM factors to convert hepatocytes intofunctional neurons, providing the first proof that an endodermalcell can be converted into an ectodermal cell (Marro et al.,2011). Renal epithelial cells from urine can be directly convertedinto neurons via lentivirus-mediated expression of BAM factorsplus NeuroD1 and c-Myc (Zhang S. Z. et al., 2016). Mitchellet al. (2014) showed the Oct4 expression alone was enough togenerate NPCs from somatic cells. These are only a few examplesof the variety of transcription factors used to generate neurons(Mitchell et al., 2014).

MicroRNA-Mediated Direct ConversionSeveral groups have also achieved direct conversion viamicroRNAs, although concomitant expression of neural lineage-specific transcription factors is required to fully mature these iNs.Coexpression of miR-9/9∗ and miR-124 is sufficient to directlyconvert human fibroblasts into neurons (Yoo et al., 2011). Whileinitial conversion efficiency using these microRNAs was very low,efficiency increased with the addition of the transcription factorsNeuroD2, Ascl1, and Myt1. Furthermore, striatal medium spinyneurons ormotor neurons can also be generated by co-expressionof miR-9/9∗ and miR-124 in addition to the transcription factorsBCL11B (CTIP12), DLX1, DLX2, and MYT1L or ISL1 andLHX3, respectively (Victor et al., 2014; Richner et al., 2015;Abernathy et al., 2017). Huh et al. also showed that directconversion using this protocol preserves age-related epigeneticand cellular signatures from the donor, similar to establishedtranscription factor-only methods (Huh et al., 2016). Primarydermal human fibroblasts can also be converted into functionalneurons by expression of miR-124 in addition to transcriptionfactors Brn2 and Myt1 (Ambasudhan et al., 2011). An additionalmethod reported by Zhou et al. (2014) showed that depletionof p53 by zinc finger nuclease (ZFN) technology is sufficient toconvert fibroblasts into neurons, oligodendrocytes, or astrocytes,depending on the selection media used (Zhou et al., 2014). It has

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not yet been established whether p53 depletion generates neuronswhich recapitulate disease pathology.

Similar to microRNA-mediated approaches, expression ofshort interfering RNA (siRNA) inhibiting the polypyrimidine-tract binding (PTB) protein has been shown to be sufficient fortransdifferentiation (Xue et al., 2013). The suppression of PTBallows for expression of various neuronal genes, including theneuron specific microRNA-124. In non-neuronal cells, the PTBprotein inhibits microRNAs which act on various componentsof the RE1-silencing transcription factor complex. Relief of thisPTB-protein inhibition leads to activation of an array of neuronallineage-specific genes, thereby promoting neuronal induction.

Lau et al. report using a self-regulating viral vector whichexpresses BAM factors and has target sequences for microRNA-124. Once the cells have been converted into neurons, they willexpress miR-124, and then downregulate the transgenes (Lauet al., 2014). The microRNA-mediated downregulation of theneural conversion genes allow for more complete functionalmaturation in culture.

MicroRNAs, with their ability to both promote expression oflineage specific genes and suppress expression after conversion,offer a path to fine-tune conversion to best replicate endogenousexpression of cell-type specific genes and more accuratelyrecreate their effect on disease biology in the patient.This benefit must be weighed against the fact that mostmicroRNAs have a variety of target genes, and overexpressionmay cause unintended dysregulation of disease-relevantgenes.

Small Molecule-Mediated DirectConversionIn addition to transcription factors, small molecules can converthuman fibroblasts into functional neurons without the additionof exogenous genetic factors. The resulting neurons are calledchemically induced neurons (CiNs). Small molecules have beenshown to increase efficiency of conversions when combinedwith transcription factors (Ladewig et al., 2012; Liu et al., 2013;Lee et al., 2015; Mertens et al., 2015; Shi et al., 2016). SMAD,GSK3β, and ALK inhibitors, alongside cyclic AMP signalingenhancers, are common betweenCiN and iPSC differentiation. Inan early study, (Ladewig et al., 2012) combined Ascl1 and Ngn2with the small molecules SB-431542, noggin, and CHIR99021 (aGSK3β inhibitor). Reports of purely small molecule conversionsinclude a combination of forskolin, isoxazole 9 (an inducer ofneural stem cell differentiation), CHIR99021 and I-BET151 (aBET bromodomain inhibitor), which can convert fibroblasts intofunctional neurons that express pan-neuronal markers (Li X.et al., 2015), and a small molecule cocktail called VCR, consistingof Valproic acid, CHIR99021 and Repsox, which is capable ofinducing NPCs (Cheng et al., 2014). Hu et al. expanded VCRand generated human CiNs using the cocktail named VCRFSGY,which consisted of Valproic acid, CHIR99021, Repsox, Forskolin,SP600125 (a JNK inhibitor) GO6983 (a PKC inhibitor) andY-27632 (a ROCK inhibitor) (Hu et al., 2015). These hCiNsshowed some spontaneous electrical activity and a train of actionpotentials when stimulated.

DIRECT CONVERSION CELL TYPES

Direct conversion has generated nearly as wide a variety ofCNS cell types as have stem cell differentiation approaches.Proneural transcription factors without subtype specificationlargely result in glutamatergic and GABA-ergic iNs (Vierbuchenet al., 2010; Wang et al., 2014; Mertens et al., 2015). Success ofspecific cell type generation by direct conversion techniques arelargely based on expression of cell type-specific markers, but thequestion remains if the final cell type generated mimics bonafide neuron subtypes. As in iPSC models, cell type confidencecan be increased by considering subtype-specific morphology,gene expression, and electrophysiology in concert with markerproteins and in comparison to individual cell types of adulthuman tissue.

Direct Brain-Native Neuronal SubtypeConversionInduced neural progenitor cells (iNPCs) can be generatedvia expression of pluripotency factors in possible combinationwith small molecules (Kim et al., 2011a; Mitchell et al., 2014;Lee et al., 2015; Hou et al., 2017). iNPCs are not post-mitotic, and can differentiate into various neuronal subtypes,oligodendrocytes, and astrocytes in vivo and in vitro. Pro-neural and serotonergic neuron-specific transcription factors,in addition to small molecules, can generate induced neuronsthat have characteristics of native serotonergic neurons suchas release of serotonin, response to SSRIs, increased expressionof specific serotonergic genes, and firing of spontaneousaction potentials (Vadodaria et al., 2016; Xu et al., 2016).Dopaminergic neurons are of particular interest due to theirclinical relevance in Parkinson’s disease. It has been shown thatinduced DA neurons (iDAs) can be generated from humanfibroblasts via a combination of transcription factors (Caiazzoet al., 2011; Kim et al., 2011b; Pfisterer et al., 2011; Liuet al., 2012; Torper et al., 2013; Dell’Anno et al., 2014). Theresulting iDAs displayed characteristic DA uptake and release,expressed DA neuron-specific markers, and were electricallyactive.

Direct Motor Neuron ConversionTransdifferentiation can also model nervous system diseaseoutside of the brain. The small molecules Forskolin andDorsomorphin, in addition to the transcription factorsNeurogenin2 and SOX1, can generate human inducedcholinergic neurons with mature electrophysiological properties,motor neuron-like features, and morphology (Liu et al., 2013).Human induced motor neurons (hiMNs), which form functionalNMJs when cultured with primary mouse skeletal myotubes, canbe generated via expression of Ngn2, SOX11, ISL1, and LHX3,in addition to the small molecules forskolin and dorsomorphin(Liu et al., 2016). Son et al. utilized a transcription factor-only (BAM factors plus motor neuron-specific transcriptionfactors Lhx3, Hb9, and Isl1, and NeuroD1) approach togenerate functional human spinal motor neurons (Son et al.,2011).

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Direct Sensory Neuron ConversionInduced sensory neurons can be generated by the transientco-expression of Brn3a with either Ngn1 or Ngn2 andexhibit properties of endogenous sensory neurons such asgene expression, Trk receptor expression, response to ligands,and morphology (Blanchard et al., 2015). Nociceptive sensoryneurons with nociceptor-specific functional receptors can beconverted from fibroblasts using BAM factors in addition to Isl2,Ngn1, and Klf7 (Wainger et al., 2015) or via OCT4 and a smallmolecule cocktail which inhibits SMAD and GSK-3β signaling(Lee et al., 2015).

Direct Glial ConversionDirect conversion can also produce glia. The transcription factorsNFIA, NFIB, and SOX9 can convert native brain embryonic andpostnatal mouse fibroblasts into astrocytes (iAstrocytes) (Caiazzoet al., 2015). Induced oligodendrocyte precursor cells (iOPCs)can be generated frommouse and rat fibroblasts by direct lineageconversion using transcription factors such as SOX10, Olig2, andZfp536, Nkx6.2, Egr2 (Najm et al., 2013; Yang N. et al., 2013;Mazzara et al., 2017). These iOPCs can differentiate in vivo andmyelinate sections of axons that are not myelinated. To date,there have been no reports of direct conversion of somatic cellsinto microglia. Further development of glial cell protocols will berequired to simulate non-cell autonomous neurological disease.

DIRECT CONVERSION MODELS OFDISEASE

The diversity of conversion methods and sub-type specific fatesgives researchers considerable flexibility for modeling disease.Disease states have been repeatedly shown not to impedeconversion into neurons. The use of iNs in studying disease isstill new and somewhat limited (Figure 2).

Amyotrophic Lateral SclerosisLim et al. generated iNeurons via the inhibition of PTB methodto investigate three mutations in the nuclear localization signalregion of the gene Fused in Sarcoma (FUS), a causative ALSgene (Lim et al., 2016). The ALS iNeurons showed increasedcytoplasmic FUS localization when compared to controls.Another report studying mechanisms of mutant FUS generatedhiMNs converted from the fibroblasts of three ALS patientswith known FUS mutations. FUS was mislocalized into thecytoplasm in all cases; however, only one of the three ALSpatients used in this study had a FUSmutation known to increasecytoplasmic retention of FUS (Liu et al., 2016), emphasizing thatthe use of patient-derived DC models aides in the discovery ofnovel disease mechanisms. These FUS ALS hiMNs also showeddramatically reduced electrical activity, failed to form functionalNMJs when co-cultured with primary mouse skeletal muscles,had shrunken somas, and were more susceptible to cell deathwhen compared to controls. Control iMNs co-cultured with gliafrom SOD1G93A mice undergo more cell death than those co-cultured with control glia, replicating previously reported non-cell autonomous effects (Son et al., 2011). Similarly, inducedNPCs from one patient with a SOD1A4V mutation and three

patients with the C9orf72 repeat expansion were differentiatedinto iAstrocytes and were found to be toxic to co-culturedprimary mouse neurons (Meyer et al., 2014). iNs convertedfrom C9orf72 ALS patients by PTB inhibition showed RNA foci,poly(GP) inclusions and poly(PR) inclusions (Su et al., 2014).These reports suggest that DC models can replicate pathologicalhallmarks of multiple ALS subtypes in ways similar to iPSC-derived models.

Alzheimer’s DiseaseCiNs from four Alzheimer’s disease patients with mutations inAPP (V717I) or PSEN1 (I167del, A434T, or S169del) showedhigher extracellular levels of Aβ40 and Aβ42 when comparedto controls. iNs from AD patients also showed an increasein total tau and phosphorylated tau (Hu et al., 2015). Similarresults regarding extracellular levels of Aβ40 and Aβ42 havebeen reported in induced NPCs differentiated into neurons fromthree AD patients, one with APOE4/E4 mutation and two withmutations in the PSEN1 gene (Hou et al., 2017).

Huntington’s DiseaseLiu et al. (2014) investigated the use of induced neurontechnology to directly convert human fibroblasts from patientswith Huntington’s disease. Human fibroblasts from HD patientswere converted by inhibition of PTB (Liu et al., 2014). The HDiNs exhibited nuclear mutant-Htt inclusions, abnormal neuriticbranching, and lower cell survival. In another study, iNPCswere generated from HD patient fibroblasts and then furtherdifferentiated into neurons which exhibited higher vulnerabilityto DNA damage than controls (Hou et al., 2017).

Spinal Muscular AtrophySpinal muscular atrophy (SMA) patient fibroblasts convertedinto hiMNs saw reduction in neurite growth rate and, after 60days in vitro, degeneration of the neurites (Zhang et al., 2017).Induced neurons from patients with mutations in the PTEN-induced putative kinase 1 (PINK1) pathway, a genetic risk factorfor Parkinson’s disease, show reduction of downstream targets ofthis pathway which are involved in regulation of mitochondrialquality control (Puschmann et al., 2017).

Direct conversion cells have demonstrated the ability tomimicdisease pathology and physiology in a number of neurologicaldisorders (Figure 2). What has not been established yet iswhether or not the retention of epigenetic signatures allowsDC models to recreate disease pathology more accurately thanrejuvenated iPSC models and whether cells generated from thesame patient using both differentiation methods would displaydifferent disease phenotypes.

CONCLUDING REMARKS

Patient-derived CNS models have expanded rapidly in the pastdecade. A variety of adult somatic cells can be reprogrammedinto iPSC’s and differentiated or directly converted into the cellsnecessary to model disease, test novel drug candidates, and beused for cell transplantations. As researchers today design newstudies of familial and sporadic neurological conditions, they

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must carefully consider whichmodel andmethodmost efficientlyanswers their scientific question (Figure 3).

Patient-derived CNS models are time intensive. While directconversion decreases the time to generate neurons from somaticsample collection due to the lack of reprogramming, the timeit takes to differentiate and mature functional neurons fromfibroblasts or iPSCs is similar. iPSC-based models offer moreefficient production of cells than direct conversion because cellsare infinitely expandable in both the iPSC stage and the NPCstage. While the yield of the desired cell type (e.g., neuronvs. astrocyte) is high, sub type (e.g., ChAT+ vs. CTIP2+)yield can vary widely between different small molecule andgrowth factor-based differentiation methods. Virally-expressedtranscription factors may increase specific cell subtype yieldin iPSC differentiations. While fibroblasts used for directconversion are infinitely expandable, the lack of an intermediateprogenitor stage creates a maximum one-to-one ratio of startingfibroblasts to desired cell type. In addition, there are reports ofdesired neuronal subtype yields of fewer than 10% (Vierbuchenet al., 2010; Pfisterer et al., 2011). Given the time and expense ofgenerating the cells, this low yield must be weighed against thescientific benefits of aging retention in the model.

One application for patient-derived cells which has not beenaddressed at length in this review, but has been recently reviewedbyMarsh and Blurton-Jones (2017), Czarzasta et al. (2017),Wanget al. (2017), and Smith et al. (2017), is the ability to implantiPSC-derived or directly converted NPCs and differentiate themin vivo to perform neurotrophic roles or replace cells lost throughdegeneration.

While comparative analyses of epigenetics and geneexpression (particularly age-related genes) in iNeurons andiPSNs have demonstrated the loss of aging signatures in

reprogramming (Mertens et al., 2015; Huh et al., 2016), theeffects of these expression changes on the ability to model diseaseis poorly understood. Whether or not certain disease hallmarks,including proteinopathies, morphological and functionalchanges, and differences in survival in response to stressors willmanifest differently when epigenetic signatures are preservedremains to be seen. A recent comparison of methylation of theC9orf72 transcript in repeat expanded eSCs and iPSCs suggeststhat toxic RNA transcription, and thus presumably RAN DPRtranslation, is attenuated in iPSCs and may suppress the rolethese mechanisms play in disease (Cohen-Hadad et al., 2016).This supports the argument that reprogramming will have aneffect on disease pathology.

The clonal nature of iPSCs allows for the generation ofCRISPR-Cas9 correction of disease-causing mutations, whichcan help establish whether certain pathologies observed inthe model are attributable to the mutation and provides anisogenic control. This technique is not currently available indirect conversion; however, there may be some advantage togenerating model neurons from a genetically and epigeneticallyheterogeneous population of somatic cells rather than fromcopies of a single iPSC clone, especially given the heterogeneityof disease pathology between cells in patient tissue.

Both direct conversion and iPSC differentiation haveproduced glia and several neuronal subtypes. Specificdifferentiation methods, including transcription factor choice,small molecule and growth factor choice, and plating methodshave been and will continue to be adjusted to create a higheryield of desired cell types in less time. While, due to its earlierdiscovery, iPSC differentiation technology is currently betterexplored and developed, it is likely that iNeurons and otherdirect conversion cell types will be equally fine-tuned in the near

FIGURE 3 | Key considerations for choosing iPSCs or DC to model neurological disease.

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future. With either method, care should be taken in regards to thetrue identity of generated cell types, given the extensive cellularconversion these cells undergo. Genetic and proteomic analysesof differentiated or converted cell types, along with comparisonsto postmortem cell-type specific analyses, can address some ofthese concerns.

The extensive reports of disease pathologies and pathogenesissummarized in this review suggest that, despite the limitationsof in vitro human cell culture models, they are capable ofrecreating disease-specific pathologies. In addition, new diseasepathways have been discovered using patient-derived cell culturemodels which were then validated in postmortem patient tissue,such as the presence of nucleocytoplasmic transport deficits andnuclear pore dysfunction in ALS, FTD, HD and, likely, in otherneurodegenerative diseases. It is therefore likely that other noveldisease mechanisms will be revealed using these human cellculture models.

Patient-derived cells offer hope for an endogenous, humanmodel of neurological disease, especially for sporadic cases whichcannot be modeled any other way, and provide a platform forrapid therapeutic discoveries that are both personalized andclinically translatable.

AUTHOR CONTRIBUTIONS

LG, AS, AN, and RS: conceptualized and wrote themanuscript.

ACKNOWLEDGMENTS

This work was support by the NIH (RO1 NS085207), theMuscular Dystrophy Association (MDA 348086), the ALSAssociation (ALSA), the Robert Packard Center for ALS Researchand the Barrow Neurological Foundation.

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